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This Article Abstract Full Text (PDF) All Versions of this Article: 90/6/815    most recent expphysiol.2005.031435v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Macdonald, W. A Articles by Nielsen, O. B Search for Related Content PubMed PubMed Citation Articles by Macdonald, W. A Articles by Nielsen, O. B Related Collections Muscle

¹ Institute of Physiology and Biophysics, University of Aarhus, Århus, Denmark

	Abstract

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In skeletal muscle, the intracellular recording of trains of action potentials is difficult owing to the movement of the muscle upon stimulation. A potential tool for the removal of muscle movement is the cross-bridge cycle blocker, N-benzyl-p-toluene sulphonamide (BTS), although the effects of BTS on the passive and active membrane properties of intact muscle fibres are not known. Rat extensor digitorum longus (EDL) muscle was used to show that 50 µM BTS reduced tetanic force to 10% of control force, without markedly altering muscle excitability. Incubation with BTS did not alter intracellular K⁺ content or Na⁺–K⁺ pump activity, but produced minor decreases in intracellular Na⁺ content (7%), resting ²²Na⁺ influx (14%) and excitation-induced ²²Na⁺ influx (29%). Despite these alterations to Na⁺ fluxes, BTS did not impair muscle excitability, since membrane conductance, resting membrane potential (RMP), rheobase current and the amplitude, overshoot and maximum rate of depolarization of the action potential were all unaltered. However, BTS did induce a small (8%) decrease in the maximum rate of repolarization of the action potential and an increase in the refractory period. The minor effects of BTS on muscle membrane properties did not compromise the ability of the muscle to propagate action potentials, even during tetanic stimulation. In conclusion, BTS can be used successfully to reduce contractility, allowing the intracellular recording of action potentials during both twitch and tetanic contraction of nerve-stimulated muscle, thus making it an excellent tool for the study of electrophysiology in fast-twitch skeletal muscle.

(Received 23 June 2005; accepted after revision 15 July 2005; first published online 19 July 2005)
Corresponding author W. Macdonald: Institute of Physiology and Biophysics, University of Aarhus, DK-8000, Århus C, Denmark. Email: wmd{at}fi.au.dk

	Introduction

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The excitation–contraction coupling (E–C coupling) of mammalian skeletal muscle fibres involves the following steps in muscle activation: (1) the generation of a muscular action potential at the neuromuscular junction; (2) action potential propagation across the sarcolemma and into the T-tubular system; (3) action potential-induced Ca²⁺ release from the sarcoplasmic reticulum (SR); and (4) activation and contraction of the contractile filaments. The investigation into many aspects of skeletal muscle E–C coupling is, however, hampered by the contraction of the muscle itself. In particular, determination of the action potential component of E–C coupling with intracellular electrodes would be greatly facilitated if the movement of the muscle, triggered by the action potential, were to be removed or inhibited. This is important because contraction of the muscle results in either breaking of the microelectrode that is inserted into the muscle fibre for the recording of action potentials, or ejection of the electrode from the fibre, thus preventing further recordings.

Conventionally, inhibition of skeletal muscle contractility for electrophysiological studies has been achieved through a number of methods, including the use of hypertonic bathing solutions (Chandler et al. 1976; Balog et al. 1994), or stretching of the muscles to reduce contractile filament overlap (Melzer et al. 1986; Cairns et al. 2003), or by the addition of substances that block the SR Ca²⁺ release channels, such as dantrolene (Radzyukevich et al. 2004). However, none of these methods are desirable, since they have confounding side-effects that can potentially alter muscle excitability. Hypertonic solutions result in the muscles becoming more difficult to penetrate with the electrodes (Hui & Maylie, 1991) and also alter Ca²⁺ release from the SR (Taylor et al. 1975). Stretching mammalian muscles far beyond their physiological length, to the extreme descending phase of the length–tension relationship, may cause severe damage to the muscles and can, in addition, activate stretch-activated channels (Franco & Lansman, 1990; McBride et al. 2000), potentially modifying muscle excitability. Dantrolene does not alter skeletal muscle resting membrane potential (RMP) or action potentials (Takauji et al. 1975), but does alter the amount of current required for membrane excitation and can only reduce twitch force by half (Oba & Hotta, 1978).

Recently, Cheung et al. (2002) described a new chemical tool, N-benzyl-p-toluene sulphonamide (BTS), which specifically blocks contraction by interfering with the cross-bridge cycling of the contractile filaments of fast-twitch skeletal muscle. Furthermore, BTS does not appear to alter the Ca²⁺ transient of electrically stimulated intact single frog fibres (Cheung et al. 2002) or of intact mouse extensor digitorum longus (EDL) muscle (Dentel et al. 2005), and does not affect SR Ca²⁺ pump ATP utilization in skinned toadfish swimbladder muscle fibres (Young et al. 2003). The potential of BTS as a tool in the investigation of skeletal muscle excitability critically depends on the specificity of BTS for myosin heavy chain type II isoform (MHC II) rather than some undesirable side-effect. So far, however, no extensive characterization of the effects of BTS on ion transport and electrical properties of the membrane of intact skeletal muscle has been published.

Here, we show that BTS inhibits tetanic force production in intact rat fast-twitch (EDL) muscles, without markedly altering the active or passive electrophysiological membrane properties. Consequently, BTS appears to be a promising tool for the selective blocking of contraction in fast-twitch skeletal muscles and thereby provides the opportunity to study many events that occur during E–C coupling, in particular the measurement of trains of intracellular action potentials in intact, nerve-stimulated muscle.

	Methods

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Animals, preparation and incubation of muscles

All handling and use of animals complied with Danish animal welfare regulations. Experiments were performed using muscles from Wistar rats bred in our department that were fed ad libitum and kept in a temperature-controlled environment at 21°C with a 12 h:12 h light:dark cycle. A few experiments were performed on muscles from 4-week-old animals weighing 60–75 g (EDL muscle weight 20–25 mg), but unless stated in the text, experiments were performed on muscles from 12-week-old animals weighing approximately 230 g (EDL muscle weight 90–110 mg). The animals were killed by cervical dislocation followed by decapitation, and intact EDL muscles were prepared and incubated in standard Krebs–Ringer bicarbonate buffer (KR) gassed continuously with a mixture of 95% O₂ and 5% CO₂ and containing the following (mM): 122.1 NaCl, 25.1 NaHCO₃, 2.8 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂ and 5.0 D-glucose (pH 7.4). BTS was dissolved in dimethyl-d₆ sulphoxide (DMSO) with matching DMSO (0.05%) added to controls. At this concentration, DMSO did not significantly alter any of the measured parameters when compared to controls without DMSO. It was not possible to use BTS at concentrations greater than 50 µM, since at higher concentrations a microcrystal precipitate formed upon the addition of BTS stock to the KR. Therefore, 50 µM BTS was the highest concentration studied. In preparations where the muscle was stimulated to contract by activating the motor nerve, the muscles were dissected out with approximately 10 mm of the nerve still attached. Unless stated specifically in the text all incubations took place at 30°C.

Measurements of force and compound action potentials (M-waves)

The muscles were mounted for isometric contractions in temperature-controlled chambers containing standard KR and adjusted to optimal length for force production. To determine the effects of BTS on tetanic force and compound action potentials (M-waves), the muscles were stimulated to contract tetanically (0.5 s trains, 60 Hz, 0.2 ms pulse duration every 20 min) either by applying field stimulation (12 V) across the central region of the muscle through platinum wire electrodes, or by stimulating the motor nerve using 5 mA constant current pulses. Force was measured using force displacement transducers and recorded with a chart recorder and/or digitally on a computer. The mean absolute force produced under initial control conditions was 0.31 ± 0.06 N (n = 16) in muscles from young animals and 0.97 ± 0.19 N (n = 16) in muscles from adult animals, with the results expressed as a percentage of initial force.

Unipolar M-wave signals were recorded from a silver electrode with a recording area of 0.79 mm², placed in close contact with the muscle between the innervation zone and the tendon (for details see Overgaard et al. 1999). The M-wave area was defined as the area between the baseline and the major negative peak of the M-wave trace, while the M-wave amplitude was defined as the maximal voltage of the negative peak, as previously described by Overgaard et al. (1999).

Resting membrane potential, action potentials and membrane properties

Recordings of action potentials and membrane properties were performed using a two electrode constant current technique, as previously described by Pedersen et al. (2005). Briefly, two electrodes were inserted into the same fibre, with one used for passing current into the cells (current electrode) and the other for measuring the intracellular potential (recording electrode). Resistances of microelectrodes were between 10 and 25 M. The current electrode was filled with 2 M potassium citrate and the recording electrode was filled with 3 M KCl. Both electrodes were connected to an Axoclamp-2A amplifier and recordings of membrane potential and current pulses injected in the fibre were displayed on an oscilloscope and recorded by a computer, using Signal 2.09 software (Cambridge Electronic Design, Cambridge, UK), at a minimum of 27 kHz.

To measure action potentials, the current and recording electrodes were placed approximately 0.3 mm apart in the same fibre. By injecting a depolarizing constant current pulse (190 nA, 3.0 ms) through the current electrode, an action potential was elicited and recorded by the recording electrode. RMP was measured from the baseline of the action potential. Action potential amplitude was defined as the difference between RMP and the peak potential of the action potential. Action potential overshoot was defined as the difference between the peak of the action potential and 0 mV. The maximum rates of depolarization and repolarization of the action potential were determined as the maximum and minimum, respectively, of the first differential of the action potential signal. The action potential refractory period was estimated by measuring the time between the peaks of the first two action potentials that occurred spontaneously in response to the injection of a 190 nA, 15 ms depolarizing constant current. A separate set of experiments was performed to determine the current required to elicit an action potential (rheobase current). Single depolarizing current pulses of 25 ms duration with progressively increasing strength (steps of 5 nA starting at 10 nA) were injected until an action potential was elicited. The current that elicited the action potential was regarded as the rheobase current.

Membrane properties of muscles were measured by injecting hyperpolarizing constant current pulses of 50 ms duration. The steady displacement of the membrane potential (V_m) during a constant current pulse (I) was recorded at three to five locations in each fibre (see Fig. 4A). For each fibre investigated, the three to five V_m/I ratios were plotted on a log scale against the interelectrode distance (x) on a linear scale and fitted to a two parameter exponentially decaying function (y(x) = y₀e^–bx) giving a straight line (see Fig. 4B). From fibres that showed an accurate fit (r² 0.99), the ordinate intercept (y₀) was taken as the input resistance (R_in) and the length constant () was calculated from the slope of the fitted line (b = 1/). Assuming an internal resistivity (R_i) of 180 cm (Albuquerque & Thesleff, 1968), R_in and were used to calculate the fibre diameter and specific membrane conductance, as described in detail by Pedersen et al. (2005). All fibres with a resting membrane potential more depolarized than –70 mV were disregarded.

View larger version (16K): [in this window] [in a new window]   Figure 4.  Effect of 50 µM BTS on the passive membrane properties Two intracellular microelectrodes were inserted into the same fibre. One electrode was used to pass hyperpolarizing constant current pulses and the other (recording electrode) was used to measure the membrane potential responses at 4 different locations along the fibre. A, representative traces showing the response of the membrane potential to injection of hyperpolarizing constant current pulses (30 nA) recorded by the recording electrode at the indicated interelectrode distances in a single fibre from a control and a BTS-treated muscle. B, plots of the ratios between the change in steady-state membrane potential and the amount of current injected from the representative trace in A, as a function of the interelectrode distance in the single fibres from the control () and BTS-treated muscles (•). See Table 2 for summarized data.

Na⁺ and K⁺ contents and exchange

Muscles from 4-week-old animals were used to determine Na⁺ and K⁺ shifts, since the small size of the muscles minimizes the diffusional barrier for the isotopes. The effect of BTS on the resting intracellular contents of Na⁺ and K⁺ was evaluated by comparing the contents of the two ions in resting control muscles with muscles incubated with 50 µM BTS for 90 min. For the measurement of resting Na⁺ influx, ²²Na⁺ (0.5 µCi ml^–1) was added to the incubation medium for the last 5 min of the incubation. Excitation-induced ²²Na⁺ influx was measured by adding ²²Na⁺ (2 µCi ml^–1) to contralateral pairs of muscles for the last 60 s of the incubation, during which one muscle was allowed to rest, while the contralateral muscle was excited with 0.2 ms, 12 V pulses delivered at 60 Hz for the last 5 s of the incubation. The excitation-induced Na⁺ influx was calculated for each pair of contralateral muscles by subtracting the measured ²²Na⁺ influx in the resting muscle from the ²²Na⁺ influx in the stimulated muscle.

In all experiments, the intracellular contents of Na⁺, K⁺ and ²²Na⁺ were determined at the end of the incubation by immediately transferring the muscles to ice-cold Na⁺-free Tris–sucrose buffer, in which they were washed for 4 x 15 min to remove extracellular Na⁺ ions and isotopes as previously described by Buchanan et al. (2002). To correct for the loss of Na⁺ and ²²Na⁺ during the 4 x 15 min washout, the values obtained were corrected by a factor of 1.46 (Buchanan et al. 2002). After wash, the muscles were extracted with 0.3 M trichloroacetic acid (TCA) and the supernatant taken for flame photometric determination of Na⁺ and K⁺ contents.

Previous studies have shown that ⁸⁶Rb⁺ is a reliable tracer for determination of K⁺ transport via the Na⁺–K⁺ pumps (Clausen et al. 1987). After the muscles had been incubated with 50 µM BTS for 90 min, they were preincubated for 15 min with or without 10^–3 M ouabain, followed by 10 min exposure to ⁸⁶Rb⁺ (0.5 µCi ml^–1) with or without ouabain. Finally the muscles were washed for 4 x 15 min in ice-cold Na⁺-free Tris–sucrose buffer to remove extracellular ⁸⁶Rb⁺. The activity of the Na⁺–K⁺ pumps was determined from the ouabain-sensitive ⁸⁶Rb⁺ uptake, as described by Buchanan et al. (2002).

Chemicals and isotopes

All chemicals were of analytical grade. BTS, ouabain and DMSO were obtained from Sigma-Aldrich. ²²Na⁺ (85 GBq mmol^–1) and ⁸⁶Rb⁺ (1590 GBq mmol^–1) were obtained from Amersham Biosciences UK, Amersham, UK.

Statistics

All data are expressed as means ± S.D. The statistical significance of any difference between groups was accepted at P < 0.05, as determined using two-way (treatment x time) ANOVA with repeated measures, together with post hoc analyses conducted using the Student–Newman–Keuls test, or Student's two-tailed t test for non-paired observations, where applicable.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of BTS on muscle contractility

The effect of BTS on the tetanic force produced by direct field stimulation is concentration and time dependent, as illustrated in Fig. 1. Representative traces showing the effect of 50 µM BTS on 60 Hz tetanic force in an EDL muscle from a 12-week-old animal are shown in Fig. 1A. Although depressed in maximum amplitude, the time course of the tetanic contractions was similar before and after BTS. Figure 1B shows that incubation with 20 µM BTS for 100 min significantly (P < 0.01) reduced force to 30% of initial force, which was further reduced to 20% of initial force after 160 min of incubation. Increasing the concentration of BTS to 50 µM resulted in a significantly (P < 0.01) faster and greater reduction in tetanic force, with less than 10% of initial force remaining after 100 min of incubation. There was no further significant reduction in tetanic force for any time point beyond 100 min.

View larger version (16K): [in this window] [in a new window]   Figure 1.  The effect of BTS on force development, elicited by field stimulation in intact EDL muscles Throughout the experiment, the force response was tested every 20 min by stimulating the muscles for 0.5 s at 60 Hz, using 0.2 ms pulses. A, representative trace of the tetanic force responses of a muscle from a 12-week-old animal before (control) and 20, 40, 60, 80, 100, 120, 140 and 160 min after the addition of 50 µM BTS. Summarized data are shown of the effects of 160 min incubation with 20 µM (•) and 50 µM () BTS on tetanic force in intact EDL muscles from adult 12-week-old rats (B) and young 4-week-old rats (C). Data points are means ± S.D., n = 4, with 100% force corresponding to an absolute force of 0.97 ± 0.19 and 0.31 ± 0.06 N for 12- and 4-week-old animals, respectively.

Effect of BTS on M-waves

In order to validate the use of BTS as a tool in the examination of muscle excitability in intact muscles, it was important to compare muscle excitability in control and BTS-treated muscles. Figure 2 shows simultaneous recordings of tetanic force and muscle M-waves, measured every 20 min following addition of 50 µM BTS. In the 100 min after BTS addition, tetanic force was significantly (P < 0.01) reduced to 10% of initial force (Fig. 2A). However, there was no significant concomitant alteration in either M-wave area (P = 0.97, Fig. 2B) or M-wave amplitude (P = 0.86, Fig. 2C), demonstrating that BTS does not appear to alter muscle excitability. Since, in these experiments, the muscles were stimulated via the nerve, the absence of any alteration of the M-wave characteristics with BTS (Fig. 2) also indicates that the function of the nerve was unaffected by BTS. Further evidence that the nerve was unaltered by BTS is provided by the similarity in time course of the force reduction between Fig. 1B, where the muscles were stimulated directly via field stimulation, and Fig. 2, where stimulation occurred via the nerve.

View larger version (16K): [in this window] [in a new window]   Figure 2.  Effect of BTS on muscle compound action potentials (M-waves) Simultaneous recordings of tetanic force elicited by 60 Hz nerve stimulation (A), M-wave area (B) and M-wave amplitude (C) for control () and BTS-treated muscles (•). BTS-treated muscles were exposed to 50 µM BTS from time zero. Data points are means ± S.D. of values and are normalized to pretreatment responses (n = 4). Representative traces of M-waves are indicated at the top of the figure.

To further investigate the effects of BTS on the sarcolemmal excitability, intracellular recordings of action potentials were performed in control and 50 µM BTS-treated muscles. Figure 3 shows that exposure to BTS did not markedly alter the shape of the action potential, with a more detailed analysis of action potential parameters reported in Table 1. BTS did not alter the RMP (P = 0.56) or the action potential amplitude (P = 0.13), overshoot (P = 0.13) or maximum rate of depolarization (P = 0.08). However, BTS caused a significant decrease in the maximum rate of repolarization of the action potential (8%, P < 0.01) and a significant increase in time between spontaneous action potential peaks (35%, P < 0.01), which is an indicator of the action potential refractory period. As also shown in Table 1, there was no significant difference (P = 0.63) in the rheobase current between control and BTS-treated muscles. These data indicate that 50 µM BTS does not alter the ability of the sarcolemma to fire action potentials and, although it causes a small decrease in the rate of repolarization of the action potential, it does not adversely effect sarcolemmal excitability.

View larger version (12K): [in this window] [in a new window]   Figure 3.  Representative traces illustrating the effect of 50 µM BTS of muscle action potentials Action potentials from control (A) and BTS-treated muscles (B). Action potentials were elicited by injecting a 190 nA depolarizing constant current pulse of 3 ms duration and subsequently recorded by a recording electrode inserted in the same individual fibre of the whole intact muscle preparation. BTS-treated muscles were incubated with 50 µM BTS for 30 min prior to commencement of recordings. Summarized data is shown in Table 1.

To investigate the effects of BTS on muscle membrane properties in a more quantitative manner, some membrane properties of control and 50 µM BTS-treated muscles were determined using the two electrode constant current technique. Figure 4A shows traces of the membrane responses in two single representative fibres from control and BTS-treated muscles, injected with 30 nA hyperpolarizing pulses. From such recordings, the length constant (), input resistance (R_in) and membrane conductance (G_m) were determined in accordance with the methods of Boyd & Martin (1959). Table 2 shows summarized data of these parameters and that the values of (P = 0.29), R_in (P = 0.67) and G_m (P = 0.15) were not altered by incubation of muscles with BTS. Thus, incubation with 50 µM BTS did not appear to alter the passive membrane properties of the muscles.

To evaluate the effect of BTS on the chemical gradients for Na⁺ and K⁺, small muscles from 4-week-old rats were incubated for 90 min in buffer with 50 µM BTS. As shown in Table 3, BTS had no effect (P = 0.49) on the intracellular K⁺ content of the muscles but led to a significant, although small, 7% reduction (P = 0.03) in the intracellular Na⁺ content. BTS incubation also caused a significant 14% reduction (P = 0.01) in the resting influx of ²²Na⁺, while the excitation-induced ²²Na⁺ influx was significantly reduced by 29% (P = 0.03). Other experiments, in which ⁸⁶Rb⁺ was used as a tracer for K⁺, showed that 90 min preincubation in buffer with 50 µM BTS had no effect (P = 0.70) on the ouabain-sensitive uptake of K⁺, the uptake being 272 ± 18 and 262 ± 18 nmol (g wet wt)^–1 min^–1 in control and BTS-treated muscles, respectively (n = 6).

Figure 5 shows representative traces of simultaneous recordings of force and intracellular action potentials during nerve stimulation of intact muscles. In a control muscle (Fig. 5A), the twitch force produced after a single action potential was large enough to eject the intracellular recording electrode from the muscle fibre. This occurred in 90% of the fibres tested (n = 28 fibres from 4 muscles). However, pretreatment of the muscle with 50 µM BTS for 90 min prior to commencement of recordings reduced force production sufficiently to prevent the ejection of the electrode (Fig. 5B) in all of the fibres tested (n = 29 fibres from 3 muscles). In addition, the force inhibition conferred by BTS reduced the muscle movement enough to allow recordings of the train of action potentials that occurred during a 0.5 s, 60 Hz tetanic contraction (Fig. 5C). Similar recordings were achieved in ten fibres from four individual muscles.

View larger version (28K): [in this window] [in a new window]   Figure 5.  Representative traces showing simultaneous recordings of force and action potentials during twitch and tetanic stimulation of muscles stimulated via the nerve Single action potentials and the corresponding twitch force in a control (A) and a 50 µM BTS-treated muscle (B). C shows a 60 Hz train of action potentials and the corresponding force production in a 50 µM BTS-treated muscle. Note that in A, the recorded potential changed to zero at the start of the contraction because the intracellular electrode was ejected from the muscle fibre. This did not happen in the BTS-treated muscles (B and C). For clarity, the stimulation artefacts have been removed.

	Discussion

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The ability of BTS to reduce twitch force production in fast-twitch muscle has previously been shown in skinned single fibres (Cheung et al. 2002; Young et al. 2003), in intact single fibres (Cheung et al. 2002) and in intact mouse skeletal muscle (Dentel et al. 2005). The present study is the first to investigate the inhibition of tetanic force with BTS (60 Hz, Figs 1 and 2), which is a more physiologically relevant stimulation regime, since the in vivo motor unit firing frequency in the EDL of adult rats is 40–111 Hz (Hennig & Lømo, 1985). However, a higher concentration of BTS was required to inhibit tetanic force than that previously recorded for twitch force (Cheung et al. 2002; Young et al. 2003; Dentel et al. 2005). The difference in BTS concentration required to inhibit force in intact versus single fibres is likely to be caused by diffusional limitation in larger preparations. This is further suggested by the slower time course of force inhibition observed in the present study, in the larger muscles compared to smaller muscles (Fig. 1). As shown in Figs 1 and 2, BTS could not completely abolish tetanic force production. One explanation for this may be that the residual force was produced by the small percentage of slow-twitch fibres that exist in rat EDL muscle (Bortolotto et al. 2000), since BTS is highly specific to fast-twitch muscle (Cheung et al. 2002). However, adult rat EDL muscles contain only 2% slow-twitch fibres (Bortolotto et al. 2000), and it is unlikely that they are responsible for eliciting 10% of tetanic force production. Moreover, even in studies on single fast-twitch fibres, BTS was unable to completely block force production (Young et al. 2003), suggesting that BTS cannot completely inhibit cross-bridge cycling.

The large reduction in tetanic force shown in Figs 1 and 2 was not due to any deleterious effects of BTS on muscle excitability, since Fig. 2 also shows that muscle M-wave area and amplitude were not significantly changed by 50 µM BTS. Upon closer inspection of the components of muscle excitability, neither the passive membrane properties (Fig. 4 and Table 2) nor the action potential parameters (Fig. 3 and Table 1) of the muscles were greatly altered with 50 µM BTS. There were, however, alterations in the maximal rate of repolarization of the action potential (8% reduction) and time between action potential peaks (35% longer). Two previous studies have also shown that BTS alters components of the action potentials of fast-twitch skeletal muscle. In intact single frog fibres, 100 µM BTS was shown to lengthen the refractory period, which the authors suggested was due to prolongation of the action potential (Cheung et al. 2002). Woods et al. (2004), using enzymatically isolated mouse flexor digitorum brevis fibres, also observed a prolongation in the decay phase of the action potential with 50 µM BTS. Furthermore, they found that BTS-treated fibres required a threefold larger current to hold the resting membrane potential at –90 mV when compared to controls, which they suggested was related to a BTS-induced alteration in membrane properties. Both of these studies were performed at relatively low temperatures of 16°C (Cheung et al. 2002) and 22°C (Woods et al. 2004). In the present study, 50 µM BTS caused no significant alterations to action potentials at 22°C, and only when the temperature was elevated to 30°C was a decrease in the rate of repolarization of the action potential observed (Table 1). However, the rheobase current and passive membrane properties (Table 2) were not altered at 30°C. The increase in refractory period in BTS-treated muscles would still allow muscles to be stimulated at frequencies up to 175 Hz, which is much greater than that occurring in vivo in rat EDL muscle (Hennig & Lømo, 1985). Therefore, the small alterations to action potentials with BTS at 30°C did not compromise the ability of the muscle to respond to stimulation at frequencies similar to those that would occur in vivo.

Incubation of resting muscles from 4-week-old animals with BTS led to a small reduction in the intracellular content of Na⁺ (Table 3) and the influx of ²²Na⁺, but had no effect on the ouabain-sensitive Rb⁺ uptake. This indicates that the reduction in intracellular Na⁺ was related to a lowered permeability of the muscle fibres to Na⁺, rather than to an increase in the activity of the Na⁺–K⁺ pumps. In addition, BTS also reduced the excitation-induced ²²Na⁺ influx (Table 3). The influx of ²²Na⁺ into the muscle fibres depends, in these experiments, on several parameters, including the activity of Na⁺-driven cotransport systems and the opening of Na⁺ channels, and possibly on the accessibility of the interstitial space to ²²Na⁺ from the incubation medium. It is not possible from the present study to identify which of these transport systems are responsible for the reduction in Na⁺ influx induced by BTS.

Despite the reduced ²²Na⁺ influx, the measurements of action potentials showed that, within the resolution obtained by data sampling at 27 kHz, BTS did not affect the rate of rise and the amplitude of the action potential (Table 1), indicating that the reduction in Na⁺ influx induced by BTS is too small to affect the generation of action potentials.

BTS inhibits force production by altering contractile filament interactions as well as reducing the activity of the ATPase associated with the contractile apparatus (Cheung et al. 2002; Shaw et al. 2003). In contract, the SR Ca²⁺ transient is unaltered by BTS (Cheung et al. 2002; Dentel et al. 2005), suggesting that SR Ca²⁺ release, myoplasmic Ca²⁺ concentration and SR Ca²⁺ reuptake are unaffected by BTS. The present study shows that BTS does not markedly alter the excitability steps of E–C coupling that precede SR Ca²⁺ release. The mechanisms of force inhibition conferred by BTS are therefore restricted to the contractile apparatus of fast-twitch muscle, with all other components of E–C coupling unaltered.

Since BTS does not markedly alter muscle excitability, it is an ideal candidate for the prevention of the muscle movement arising during the study of components of skeletal muscle E–C coupling and in particular muscle electrophysiology. As already mentioned in the Introduction, the techniques previously used to reduce muscle movement when recording action potentials with intracellular electrodes have some undesirable characteristics. In the present study, we also used a two electrode technique (Albuquerque & Thesleff, 1968; McArdle et al. 1980) for intracellular electrode recordings. Since only one fibre in the muscle is stimulated with this technique, the movement of the whole muscle is markedly reduced and limited to the specific fibre impaled by the electrodes. Furthermore, this technique allows for the determination of parameters such as rheobase current. However, even using this technique we often observed that electrodes were ejected from the fibres of control muscles upon injection of current via the current electrode. One further disadvantage of this technique is that it does not allow for nerve stimulation of the muscle, preventing the entire muscle–nerve preparation from being studied as a single functional unit.

Despite the small residual force that remains in BTS-treated muscles (Figs 1 and 2), the contraction, and therefore muscle movement, is sufficiently reduced to prevent the ejection of the intracellular electrodes from the muscle fibres during stimulation. This was clearly demonstrated in Fig. 5, where nerve stimulation of muscles producing twitch force ejected the intracellular electrode from the muscle fibre in control muscles, but not in BTS-treated muscles. Even during a 60 Hz tetanic contraction lasting 0.5 s, the intracellular electrode remained in the muscle fibre of BTS-treated muscles. This was not possible in muscles without BTS. Since BTS does not significantly alter the M-wave characteristics when the muscles are stimulated indirectly via the nerve, and since the time course of the force reduction in muscles stimulated directly or indirectly is the same, it is assumed that the motor nerve is unaffected by BTS. This is of great advantage, since it allows the use of nerve stimulation for studying the excitation components of muscle contraction (motor nerve, motor endplate, sarcolemma and T-system) as a single excitatory entity. Activation via the nerve also allows the transmission of substances that would normally be present in the motor endplate together with acetylcholine, giving a more accurate overall picture of muscle excitation. The inhibitory effect of BTS is primarily on the contractile apparatus, and BTS does not greatly affect the Na⁺ and K⁺ fluxes associated with normal E–C coupling and, unlike dantrolene, does not alter the Ca²⁺ transient. BTS is an obvious choice for the removal of skeletal muscle movement in studies involving fluorescent dyes and imaging, in particular myoplasmic Ca²⁺ imaging during muscle contraction.

In conclusion, at 50 µM, BTS has no marked effects on any of the excitatory events that occur in skeletal muscle E–C coupling and appears to be an excellent tool to use for inhibiting force production, and thus the removal of the muscle movement. Furthermore, BTS is effective over both the range of temperatures and the ages of animals often used during in vitro skeletal muscle experiments.

	References

Top Abstract Introduction Methods Results Discussion References

 
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	Acknowledgements

 
We thank Ann-Charlotte Andersen, Marianne Stürup Johansen, Tove Lindahl Andersen and Vibeke Uhre for skilled technical assistance. Also, we thank Dr John A. Flatman for useful discussions and technical assistance. This study was supported by grants from Aarhus Universitets Forskningsfond, The Lundbeck Foundation, The Danish Medical Research Council (22-02-0188), and a PhD grant for Thomas Holm Pedersen from the Faculty of Medical Sciences, University of Aarhus.

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